Project Summary/Abstract Liver disease is a growing clinical problem in the United States and worldwide affecting 30 million Americans resulting in 750,000 hospitalizations and 36,000 deaths yearly. Unfortunately, unlike other common diseases such as cardiovascular diseases, the incidence of liver disease and its associated complications are increasing. Given the steady rise of patients with liver disease, the demand for liver transplantation has continued to increase while the supply of organs has remained unchanged creating a pressing need to address this organ scarcity. Proposed alternatives to organ transplantation are the transplantation of cells but human hepatocytes are similarly difficult to source. As a result, there are currently no alternatives for obtaining robust functional hepatocytes which is significantly hampering scientific study and the development of more effective clinical therapies; our work addresses this significant research and clinical gap. Pluripotent stem cell derived hepatocyte-like cells are often cited as a potential alternative cell source for organ transplants and as a platform for pharmaceutical and scientific study. However, the current inability to derive robust and fully mature hepatocytes limits the clinical and scientific utility of currently produced cells. Pluripotent stem cell fate and hepatocyte maturation is determined by the complex array of internal signals and external cues from the microenvironment. Unfortunately our current differentiation platforms do not mimic the cellular, extracellular matrix (ECM), and 3D complexities of the in vivo hepatic microenvironment and therefore the role of ECM-cell and cell-cell interactions in the differentiation process remains largely unknown. Our central hypothesis is that the 3D microenvironment and subsequent cellular interactions (e.g. cell-ECM and cell-cell interactions) dictates cell fate decisions and it is through these factors by which fetal cell types mature into terminally differentiated cell types. In recent work our group has developed robust tools to systematically and efficiently manipulate the 3D microenvironment. The central goal of this proposal is to identify and examine important inductive cellular interactions for directed pluripotent stem cell differentiation in vitro to enable the generation of phenotypically and functionally mature hepatocytes. We will systematically examine and identify the role cell-cell and cell- ECM interactions play in regulating the fetal to adult hepatocyte transition and will incorporate these findings into our current differentiation and tissue engineered constructs. These optimized 3D multicellular spheroids platforms will enable scientific study and the development of more effective clinical therapies.